Successful DNA amplification relies on the integrity of the DNA template but aged samples or samples stored under sub-optimal conditions often results in failed amplification reactions.
DNA both in the living cells and isolated in vitro is subject to many chemical alterations (a fact often forgotten in the excitement of being able to do DNA sequencing on dried and/or frozen specimens). If the genetic information encoded in the DNA is to remain uncorrupted, any chemical changes must be corrected. The recent publication of the human genome has already revealed 130 genes whose products participate in DNA repair.
Agents that damage DNA include: ionizing radiation such as gamma rays and x-rays ultraviolet rays, especially the UV-C rays (˜260 nm) that are absorbed strongly by DNA but also the longer-wavelength UV-B, highly-reactive oxygen radicals produced during normal cellular respiration as well as by other biochemical pathways, many hydrocarbons, including some found in cigarette smoke, some plant and microbial products and chemicals used in chemotherapy, especially chemotherapy of cancers
All four of the bases in DNA (A, T, C, G) can be covalently modified at various positions. One of the most frequent is deamination, resulting for example, in a C being converted to a U. Mismatches of the normal bases because of a failure of proofreading during DNA replication. A common example is incorporation of the pyrimidine U (normally found only in RNA) instead of T.
Nicks in the DNA backbone can be limited to one of the two strands (a single-stranded break, SSB) or on both strands (a double-stranded break (DSB). Ionizing radiation is a frequent cause, but some chemicals produce breaks as well.
Covalent cross-linkages can be formed between bases on the same DNA strand (“intra-strand”) or on the opposite strand (“inter-strand”).
Various types of DNA damage can occur during improper storage, aging, freeze-thawing, or exposure to acid, heat or light. This will adversely affect the performance of the DNA as template in PCR because the damage will block the progression of the DNA polymerase.
Enzyme reagents for use in the modification and repair of degraded DNA are available commercially, for example from New England BioLabs (neb.com/nebecomm/products/category12.asp#16). However, only recently has attention been focused on combining such enzyme reagents for the purpose of repairing DNA and improving the DNA template integrity for subsequent amplification reactions. For example, Restorase™ (Sigma Aldrich) has been demonstrated to repair AP sites, but not other types of DNA damage such as backbone nicks.
There remains a need for combinations of enzymes to effect repair of the many different classes of DNA damage for subsequent amplification reactions, particularly since alternative amplification methods to PCR are gaining prominence. One particularly useful method is whole genome amplification by multiple displacement amplification (MDA). MDA has been developed for the whole genome amplification of human DNA samples. MDA relies on priming the genomic DNA with exonuclease resistant random primers and the use of a strand displacing DNA polymerase such as Φ29 DNA polymerase. Φ29 DNA polymerase is highly processive, strand displacing and has a very low error rate of 1 in 106-107 nucleotides compared to an error rate of 3 in 104 for native Taq polymerase and 1.6 in 106 for Pfu polymerase. Due to the high processivity and strand displacing properties of Φ29 DNA polymerase the MDA reaction is performed under isothermal conditions at 30° C. The combination of a highly processive enzyme and random priming makes MDA amplification ideal approach for the amplification of DNA where there is currently insufficient material for use standard DNA typing technologies.
Other such alternatives to PCR amplification include isothermal helicase-dependent amplification, LATE-PCR assymmetric amplification, and single primer isothermal linear amplification.
Endonuclease VIII from E. coli acts as both an N-glycosylase and an AP-lyase. The N-glycosylase activity releases degraded pyrimidines from double-stranded DNA, generating an AP site. The AP-lyase activity cleaves 3′ to the AP site leaving a 5′ phosphate and a 3′ phosphate. Degraded bases recognized and removed by Endonuclease VIII include urea, 5,6-dihydroxythymine, thymine glycol, 5-hydroxy-5-methylhydantoin, uracil glycol, 6-hydroxy-5,6-dihydrothymine and methyltartronylurea. While Endonuclease VIII is similar to Endonuclease III, Endonuclease VIII has β and δ lyase activity while Endonuclease III has β lyase activity (New England BioLabs—neb.com/nebecomm/products/productM0299.asp).
Fpg (formamidopyrimidine [fapy]-DNA glycosylase) (also known as 8-oxoguanine DNA glycosylase) acts both as an N-glycosylase and an AP-lyase. The N-glycosylase activity releases degraded purines from double stranded DNA, generating an apurinic (AP site). The AP-lyase activity cleaves both 3′ and 5′ to the AP site thereby removing the AP site and leaving a 1 base gap. Some of the degraded bases recognized and removed by Fpg include 7,8-dihydro-8-oxoguanine (8-oxoguanine), 8-oxoadenine, fapy-guanine, methyl-fapy-guanine, fapy-adenine, aflatoxin B1-fapy-guanine, 5-hydroxy-cytosine and 5-hydroxy-uracil (New England BioLabs—neb.com/nebecomm/products/productM0240.asp).
USER (Uracil-Specific Excision Reagent) enzyme generates a single nucleotide gap at the location of a uracil. USER Enzyme is a mixture of Uracil DNA glycosylase (UDG) and the DNA glycosylase-lyase Endonuclease VIII. UDG catalyses the excision of a uracil base, forming an abasic (apyrimidinic) site while leaving the phosphodiester backbone intact. The lyase activity of Endonuclease VIII breaks the phosphodiester backbone at the 3′ and 5′ sides of the abasic site so that base-free deoxyribose is released (New England BioLabs—neb.com/nebecomm/products/productM5505.asp).
Endonuclease IV can act on a variety of oxidative damage in DNA. The enzyme is apurinic/apyrimidinic (AP) endonuclease that will hydrolyze intact AP sites in DNA. AP sites are cleaved at the first phosphodiester bond that is 5′ to the lesion leaving a hydroxyl group at the 3′ terminus and a deoxyribose 5′-phosphate at the 5′ terminus. The enzyme also has a 3′-diesterase activity and can release phosphoglycoaldehyde, intact deoxyribose 5-phosphate and phosphate from the 3′ end of DNA (New England BioLabs—neb.com/nebecomm/products/productM0304.asp).
DNA Polymerase I, Large (Klenow) Fragment is a proteolytic product of E. coli DNA Polymerase I which retains polymerization and 3′→5′ exonuclease activity, but has lost 5′→3′ exonuclease activity. Klenow retains the polymerization fidelity of the holoenzyme without degrading 5′ termini. This enzyme is used in applications such as filling-in of 5′ overhangs to form blunt ends, removal of 3′ overhangs to form blunt ends, and second strand cDNA synthesis (New England BioLabs-neb.com/nebecomm/products/productM0210.asp).
T4 polynucleotide kinase catalyzes the transfer and exchange of a phosphate group from the γ position of ATP to the 5′-hydroxyl terminus of polynucleotides (double- and single-stranded DNA and RNA) and nucleoside 3′-monophosphates and also catalyzes the removal of 3′-phosphoryl groups from 3′-phosphoryl polynucleotides, deoxynucleoside 3′-monophosphates and deoxynucleoside 3′-diphosphates (New England BioLabs—neb.com/nebecomm/products/productM0201.asp).
T4 DNA ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5′ phosphate and 3′ hydroxyl termini in duplex DNA or RNA. This enzyme will join blunt end and cohesive end termini as well as repair single stranded nicks in duplex DNA, RNA or DNA/RNA hybrids (New England BioLabs—neb.com/nebecomm/products/productM0202.asp).
The present invention satisfies the need for providing, inter alio, combinations of DNA repair enzymes that are useful for restoring the integrity of degraded DNA templates so that effective and efficient amplification can be obtained and subsequent analyses carried out. Examples of such subsequent analyses include, but are not limited to, methods for identification of bacterial and viral bioagents for biodefense, diagnostics and forensics investigations which are disclosed and claimed in U.S. patent application Ser. Nos. 09/798,007, 10/660,122, 10/728,486, 10/405,756, 10/660,998 and 10/807,019, each of which is commonly owned and incorporated herein by reference in its entirety.